Abstract
Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.
Highlights
Stressful environments accelerate the formation of isoaspartyl residues in proteins, which detrimentally affect protein structure and function
We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PROTEIN LISOASPARTYL METHYLTRANSFERASE (PIMT) activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival
We have shown the detrimental effects of stress induced isoAsp accumulation in protein functions and the consequent adverse effect on plant growth and survivability under heat and oxidative stress environments, and the protective role of PIMT in mitigating such detrimental effects
Summary
Heat and oxidative stresses induce isoAsp accumulation in proteins in Arabidopsis. As a first step toward understanding the importance of the PIMT activity in plant stress tolerance, we surveyed isoAsp accumulation in proteins at the seedling and mature plant stage of Arabidopsis under standard growth conditions as well as under heat and oxidative stresses. Like ROS, MDA content was significantly increased in RNAi lines and markedly reduced in overexpressing lines as compared with the WT plants under heat and oxidative stress conditions (Fig. 4d and Fig. S8c) The results of these experiments suggest that PIMT somehow plays an important role in restricting ROS accumulation under stressful environments. Compromised activities of CAT and SOD were observed in AtPIMT1 and AtPIMT2 RNAi lines, whereas enhanced CAT and SOD activities were seen in PIMT-overexpressing lines relative to that of the WT plants, under stressful environments (Fig. 4, e and f) These results suggest that PIMT plays an important role in maintaining the functionality of antioxidant enzymes, possibly by preventing them from isoAsp-mediated damage, under stressful conditions, thereby preventing ROS accumulation in plants during stressful environments. Our results strongly suggest that PIMT plays an important role in plant stress tolerance by maintaining ROS homeostasis during stress
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